Identification and isolation of transitional cell carcinoma stem cells

ABSTRACT

Transitional cell carcinoma stem cells (TCCSC) are identified. The cells can be prospectively isolated or identified from primary tumor samples, and are shown to possess the unique properties of cancer stem cells in functional assays for cancer stem cell self-renewal and differentiation, and in cancer diagnosis.

BACKGROUND OF THE INVENTION

Bladder cancer is the second most common urological malignancy in theUnited States, which accounts for approximately 13,000 deaths and 61,000new cases per year. Ninety percent of bladder malignancies areclassified as transitional cell carcinomas (TCCs), which originate fromthe transitional urothelium that is composed of 3-6 layers thick ofbasal, intermediate and multinucleate umbrella cells. Seventy percent oftumors are low grade non-invasive papillary lesions at diagnosis, whichcommonly recur, only 15-20% of which progress into muscle invasivedisease. On the other hand, ˜30% of TCCs are muscle invasive atdiagnosis. Fifty percent of patients with invasive TCCs die frommetastasis within 2 years, and the 5-year survival rate for metastaticbladder cancer is only 6%.

It has long been observed that patient bladder tumors containintratumoral heterogeneity, consisting of tumor cells with diversehistological morphologies and distinct biological properties. Primarybladder tumor cells possess differential ability for anchorageindependent growth, a property defining the transforming ability ofcancer cells. This is supported by evidence that only ˜0.7% of primarybladder tumor cells have the ability to form colonies in the classicaltwo-layered soft-agar assay, which assays for anchorage independentgrowth properties of tumor cells. In addition, bladder tumor cellsdemonstrate diverse proliferation and differentiation status, mostlysupported by histological analysis showing distinct staining patterns ofproliferation markers (i.e. Ki67 and PCNA) and keratin markers definingdifferentiation status (i.e. K5/14, K8 and K20).

Basic cancer research has focused on identifying the genetic changesthat lead to cancer. This has led to major advances in our understandingof the molecular and biochemical pathways that are involved intumorigenesis and malignant transformation. However, our understandingof the cellular biology has lagged. A large body of literature hasexamined the effects of particular mutations on the proliferation andsurvival of model cells, such as cell lines, fibroblasts and mostrecently in primary epithelial cells; however, the target cellaccumulating actual mutations remained to be elucidated.

A tumor can be viewed as an aberrant organ initiated by a tumorigeniccancer cell that acquired the capacity for indefinite proliferationthrough accumulated mutations. In this view of a tumor as an abnormalorgan, the principles of normal stem cell biology can be applied tobetter understand how tumors develop. Many observations suggest thatanalogies between normal stem cells and tumorigenic cells areappropriate. Both normal stem cells and tumorigenic cells have extensiveproliferative potential and the ability to give rise to new (normal orabnormal) tissues. Both tumors and normal tissues are composed ofheterogeneous combinations of cells, with different phenotypiccharacteristics and different proliferative potentials.

Because most tumors have a clonal origin, the original tumorigeniccancer cell gives rise to phenotypically diverse progeny, includingcancer cells with indefinite proliferative potential, as well as cancercells with limited or no proliferative potential. This suggests thattumorigenic cancer cells undergo processes that are analogous to theself-renewal and differentiation of normal stem cells. Tumorigenic cellscan be thought of as cancer stem cells that undergo an aberrant andpoorly regulated process of organogenesis analogous to what normal stemcells do. Although some of the heterogeneity in tumors arises as aresult of continuing mutagenesis, it is likely that heterogeneity alsoarises through the aberrant differentiation of cancer cells.

It is well documented that many types of tumors contain cancer cellswith heterogeneous phenotypes, reflecting aspects of the differentiationthat normally occurs in the tissues from which the tumors arise. Thevariable expression of normal differentiation markers by cancer cells ina tumor suggests that some of the heterogeneity in tumors arises as aresult of the anomalous differentiation of tumor cells. Examples of thisinclude the variable expression of myeloid markers in chronic myeloidleukaemia, the variable expression of neuronal markers within peripheralneurectodermal tumors, the variable expression of milk proteins or theestrogen receptor within breast cancer, and the differential expressionof cytokeratins in a wide spectrum of epithelial tumors includingbladder cancer.

It was first extensively documented in acute myeloid leukaemia that onlya small subset of cancer cells is responsible for the tumor-initiatingpotential and maintain the ability to self-renew. Because thedifferences in clonogenicity among the leukemia cells mirrored thedifferences in clonogenicity among normal hematopoietic cells, theclonogenic leukemic cells were described as leukemic stem cells. It hasalso been shown for solid cancers that the cells are phenotypicallyheterogeneous and that only a small proportion of cells are tumorigenicand can self-renew in vivo. Just as in the context of leukemic stemcells, these observations led to the hypothesis that only rare cancerstem cells exist in epithelial tumors.

In support of this hypothesis, recent studies have shown that, similarto leukemia and other hematologic malignancies, tumorigenic andnon-tumorigenic populations of breast cancer cells can be isolated basedon their expression of cell surface markers. In many cases of breastcancer, only a small subpopulation of cells had the ability to form newtumors. This work strongly supports the existence of CSC in breastcancer. Further evidence for the existence of cancer stem cellsoccurring in solid tumors has been found in central nervous system (CNS)malignancies. Using culture techniques similar to those used to culturenormal neuronal stem cells it has been shown that neuronal CNSmalignancies contain a small population of cancer cells that areclonogenic in vitro and initiate tumors in vivo, while the remainingcells in the tumor do not have these properties.

Stem cells are defined as cells that have the ability to perpetuatethemselves through self-renewal and to generate mature cells of aparticular tissue through differentiation. In most tissues, stem cellsare rare. As a result, stem cells must be identified prospectively andpurified carefully in order to study their properties. Perhaps the mostimportant and useful property of stem cells is that of self-renewal.Through this property, striking parallels can be found between stemcells and cancer cells: tumors may often originate from thetransformation of normal stem cells, similar signaling pathways mayregulate self-renewal in stem cells and cancer cells, and cancers maycomprise rare cells with indefinite potential for self-renewal thatdrive tumorigenesis.

The presence of cancer stem cells has profound implications for cancertherapy. At present, all of the phenotypically diverse cancer cells in atumor are treated as though they have unlimited proliferative potentialand can acquire the ability to metastasize. For many years, however, ithas been recognized that small numbers of disseminated cancer cells canbe detected at sites distant from primary tumors in patients that nevermanifest metastatic disease. One possibility is that immune surveillanceis highly effective at killing disseminated cancer cells before they canform a detectable tumor. Another possibility is that most cancer cellslack the ability to form a new tumor such, that only the disseminationof rare cancer stem cells can lead to metastatic disease. If so, thegoal of therapy must be to identify and kill this cancer stem cellpopulation.

The prospective identification and isolation of cancer stem cells willallow more efficient identification of diagnostic markers andtherapeutic targets expressed by the stem cells. Existing therapies havebeen developed largely against the bulk population of tumor cells,because the therapies are identified by their ability to shrink thetumor mass. However, because most cells within a cancer have limitedproliferative potential, an ability to shrink a tumor mainly reflects anability to kill these cells. Therapies that are more specificallydirected against cancer stem cells may result in more durable responsesand cures of metastatic tumors.

Epithelial tumors contain a mixed population of cancer cells. It hasbeen hypothesized that functional heterogeneity rather than cellularheterogeneity may account for the fact that not all of the cancer cellsin solid tumors have a similar ability to drive tumor formation.Mortality from these diseases remains high due to the development ofdistant metastasis and the emergence of therapy resistant local andregional recurrences. It is therefore essential that we develop a deeperunderstanding of the biology of this disease in order to develop moreeffective therapies.

Cancer stem cells are discussed in, for example, Pardal et al. (2003)Nat Rev Cancer 3, 895-902; Reya et al. (2001) Nature 414, 105-11; Bonnet& Dick (1997) Nat Med 3, 730-7; Al-Hajj et al. (2003) Proc Natl Acad SciUSA 100, 3983-8; Dontu et al. (2004) Breast Cancer Res 6, R605-15; Singhet al. (2004) Nature 432, 396-401.

SUMMARY OF THE INVENTION

Transitional cell carcinoma stem cells (TCCSC) are identified herein.The cells can be prospectively isolated or identified from primary tumorsamples, and are shown to possess the unique properties of cancer stemcells in functional assays for tumor initiation, cancer stem cellself-renewal and differentiation. In addition, cancer stem cells can beused as a predictor for disease progression. The TCCSC have thephenotype of being positive for expression of CD44, and retaincytokeratin markers and cellular morphology similar to normal urothelialbasal cells, expressing CK5, but not CK20.

Molecular profiling of the TCCSC shows a heterogeneity of self-renewalpathways, where the cells may differentially show increased nuclearlocalization of one or more of β-catenin; Stat3; or Bmi-1 relative to anormal counterpart cell. These data revealed significant clinicalimplications, that different subgroup of TCC patients can respond todrugs that target different sets of signaling pathways. In someembodiments of the invention, the TCCSC are classified according toself-renewal pathway, which classification is useful in drug screening,and in developing a course of therapy suitable for the patient.

In some embodiments of the invention, methods are provided forclassification or clinical staging of transitional cell carcinomasaccording to the stem cells that are present in the carcinoma, wheregreater numbers of stem cells are indicative of a more aggressive cancerphenotype. Staging is useful for prognosis and treatment. In someembodiments, a tumor sample is analyzed by histochemistry, includingimmunohistochemistry, in situ hybridization, and the like, for thepresence of cells that co-express CD44 at the cell membrane and nuclearlocalization of one or more of β-catenin; Stat3; or Bmi-1. The presenceof such cells indicates the presence of TCCSC, and allows the definitionof cancer stem cell domains in the primary tumor, as well as cells inlymph node or distant metastases.

In another embodiment of the invention, compositions of isolated TCCSCare provided. The cells are useful for experimental evaluation, and as asource of lineage and cell specific products, including mRNA speciesuseful in identifying genes specifically expressed in these cells, andas targets for the discovery of factors or molecules that can affectthem. TCCSC may be used, for example, in a method of screening acompound for an effect on the cells. This involves combining thecompound with the cell population of the invention, and then determiningany modulatory effect resulting from the compound. This may includeexamination of the cells for viability, toxicity, metabolic change, oran effect on cell function. The phenotype of TCCSC described hereinprovides a means of predicting disease progression, relapse, anddevelopment of drug resistance. Methods are also provided foradministration of therapeutic agents that target cancer stem cells.Identifying TCCSCs by phenotype and signaling pathways unique to themprovides a more specific target than conventional therapies.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. Prospective identification of a rare population of CD44 positivetumor cells in patient transitional cell carcinomas by flow cytometry.(A) FACS plot showing forward scatter versus viable cells as labeled byPI staining. (B) FACS plot showing the exclusion of intra-tumoralhematopoietic cells by the cell surface marker CD45.

FIG. 2. Development of orthotopic and ectopic xenograft models for humantransitional cell carcinoma. (A) Injection of at least 1×10⁶unfractionated patient bladder tumor cells in the bladder wall of Rag2;gamma chain double knock out mice; white arrow indicate engraftment siteof xenograft tumor, black arrow indicate the location of mouse bladder.(B) Engraftment of patient bladder tumor cells in the dermal compartmentof Rag2; gamma chain double knock out mice. (C and D) Hematoxylin andEosin staining of patient tumor reveals transitional cell histology withsquamous differentiation; black square in C indicates the area ofpatient tumor magnified in a higher power as shown in D. (E and F)Hematoxylin and Eosin staining of xenograft tumor isolated fromimmunocompromised mice revealing similar histology to original patienttumor shown in C and D; black square in E indicates the area ofxenograft tumor magnified in a higher power as shown in F.

FIG. 3. CD44 positive TCC tumor cells exhibit unique properties of CSC:enrichment for tumor-initiating potential, self-renewal anddifferentiation. (A) Analysis of primary patient TCC cells on theexpression of the cell surface receptor CD44 by flow cytometry(population indicated in black box). (B) Flow cytometry analysis showingthe purity of CD44⁺ tumor cell population after sort. (C) Analysis ofTCC xenograft tumor cell on the expression of CD44⁺ (populationindicated in black box) and lineage markers (mouse CD45, CD31 and H2 Kdantibodies). (D, E and F) Representative photographs showing the dorsalside of immunocompromised mice that were incubated with different dosesof tumor cell subpopulations (white arrows indicate injection siteswhere CD44⁺ tumor cells were incubated; blue arrows indicate injectionsites where CD44⁻ tumor cells were incubated). (G) H&E staining of axenograft tumor formed from the CD44⁺ tumor cell fraction. (H) H&Estaining of a xenograft tumor formed from the CD44⁻ tumor cell fraction.

FIG. 4. Molecular analysis of β-catenin and the MAP kinase signalingpathways in CD44 positive and CD44 negative tumor cells. (A and B)Representative confocal microscopic images of unphosphorylated β-catenin(Red) in CD44 positive tumor cells from patient bladder tumor. Bluecolor indicates DAPI nuclear staining. White arrow indicates tumor cellswith nuclear localization or activated form of β-catenin. (C) Confocalmicroscopic images of unphosphorylated β-catenin (Red) in CD44 negativetumor cells from the same patient bladder tumor. Blue arrow indicates alarge multinucleate cell with umbrella cell morphology (differentiatedcell in urothelium). (D and E) Representative confocal microscopicimages of unphosphorylated β-catenin (Green) in CD44 positive tumorcells from serially transplanted xenograft tumor. Blue color indicatesDAPI nuclear staining. White arrow indicates tumor cells with nuclearlocalization or activated form of β-catenin. (F) Confocal microscopicimages of unphosphorylated β-catenin (Green) in CD44 negative tumorcells from the same xenograft tumor. (G) Microfluidics western analysisof β-catenin protein level from CD44 positive (Red) and CD44 negative(Black) tumor cells from patient bladder tumor. The sizes of peakindicate the relative protein levels. (H) Microfluidics western analysisof MAP kinase signaling (Erk1 and Erk2 protein level) from CD44 positive(Red) and CD44 negative (Black) tumor cells from the same patientbladder tumor. The sizes of peak indicate the relative protein levels.

FIGS. 5A-5B. (A) Sectional staining of nuclear beta-catenin in subsetsof TCC basal cells at the tumor-stromal junction. (B) Nuclearbeta-catenin in migrating TCC cells.

FIG. 6. Characteristics and histology of xenograft tumors formed fromCD44+ and CD44− bladder TCC subpopulations. (A) Hematoxylin and eosin(H&E) staining of patient tumor #8 (B) H&E staining of xenograft tumorformed from patient #8. (C & E) H&E staining of lymph node from patient#8 that is infiltrated with transitional carcinoma cells. (D) Photographshowing an enlarged axillary lymph node in Rag2/gamma chain double knockout mice that was engrafted with transitional carcinoma cells frompatient #8. (F) HE staining of axillary lymph node from mousedemonstrating similar histology to transitional cells from patient #8.(H & K) Flow cytometry analysis of CD44 expression in two representativepatient tumor (#1 & 8) and respective xenograft tumors either formedfrom a pure patient CD44⁺ (Black box) or CD44⁻ (Gray box) tumor cellpopulation. Patient CD44⁺ tumor cells can form xenografts comprising ofCD44⁺ (Black box) and CD44⁻ (Gray Box) tumor cells; while patient CD44−tumor cells only form xenografts comprising of CD44− tumor cells. BlueBox indicates infiltrating mouse cells that are positive for CD45(hematopoietic cell marker), CD31 (endothelial cell marker) or H2 Kd(mouse major histocompatibility class I). (G) Photographs showing theengraftment of xenograft tumors in limiting dilution of CD44⁺ and CD44⁻tumor cells from patient #1. (I) H&E staining of a representativexenograft tumor formed from patient CD44+ tumor cells (Black Box),comprising areas of less differentiated cells (indicated by *) and areasof terminally differentiated cells (indicated by arrows). (J) H&Estaining of a representative xenograft tumor formed from patient CD44−tumor cells, primarily comprising highly keratinized, terminallydifferentiated cells (indicated by arrows).

FIG. 7. CD44+ TCC subpopulation retain cytokeratin markers and cellularmorphologies resembling normal urothelial basal cells. (A) Illustrateddiagram summarizing the relative distribution of CD44, CK5 and CK20positive bladder TCCs in a bladder cancer tissue array containing ˜300specimens. Red color indicates positive immunohistochemical staining;green indicates negative staining and grey indicates data that aremissing. Arrow indicates the subgroup of bladder TCCs expressing allthree markers (i.e. CD44, CK5 and CK20). (B) Immunohistochemicalanalysis of CD44, cytokeratin 5 (basal cell marker) and cytokeratin 20(differentiated cell marker) in serial sections of four representativebladder TCCs from group 8 (indicated by arrow), brown color indicatespositive staining. (C) Giema-Wright staining showing the cellularmorphology of fractionated CD44+ and CD44− tumor cells (patient #3).

FIG. 8. Immunohistochemical analysis in CD44+ bladder TCCs revealsheterogeneity in pathway activation of oncogenes that are alsoimplicated in self-renewal of stem cells. (A) Illustrated diagramsummarizing the relative distribution of CD44 positive bladder TCCspecimens in relation to Stat3, Bmi-1, β-catenin, Oct-4 and Nanog. Oct-4and Nanog are inactive in all bladder TCCs analyzed. (B) Representativeimmunohistochemical illustrations showing the subgroup of patientspecimens coexpressing CD44 and nuclear Stat3 in serial sections; (C)Representative immunohistochemical illustrations showing the subgroup ofpatient specimens coexpressing CD44 and nuclear Bmi-1 in serialsections; (D) Immunohistochemical analysis of CD44 and β-catenin showingdifferent combinations of co-localization. Brown color indicatespositive staining and nuclear localization of β-catenin indicatespathway activation; (E & F) Immunohistochemical analysis of Nanog andOct-4 in normal testis and seminoma as positive control (Brown colourindicates positive staining).

DETAILED DESCRIPTION OF THE EMBODIMENTS

Transitional cell carcinomas are staged by analysis of the presence ofcancer stem cells. Staging is useful for prognosis and treatment. In oneembodiment of the invention, a cystectomy sample from a transitionalcell carcinoma patient is stained with reagents specific for CD44; andoptionally a lineage panel. The analysis of staining patterns providesthe relative distribution of TCCSC, which distribution predicts thestage of carcinoma. In some embodiments, the cystectomy sample isanalyzed by histochemistry, including immunohistochemistry, and thelike, for the presence of cells that co-express CD44 at the cellmembrane and one or more of β-catenin; Stat3; or Bmi-1 in the nucleus.The cells may also be analyzed for cytokeratin expression, whereexpression of CK5 is associated with the TCCSC. The presence of suchcells indicates the presence of TCCSC, and allows the definition ofcancer stem cells in the primary tumor, as well as cells in lymph nodeor distant metastases. The cells may also be classified according to thespecific self-renewal pathway that has been activated.

In one embodiment, the patient sample is compared to a control, or astandard test value. In another embodiment, the patient sample iscompared to a pre-carcinoma sample, or to one or more time pointsthrough the course of the disease.

Samples, including tissue sections, slides, etc. containing atransitional cell carcinoma tissue, are stained with reagents specificfor markers that indicate the presence of cancer stem cells. Samples maybe frozen, embedded, present in a tissue microarray, and the like. Thereagents, e.g. antibodies, polynucleotide probes, etc. may be detectablylabeled, or may be indirectly labeled in the staining procedure. Thedata provided herein demonstrate that the number and distribution ofprogenitor cells is diagnostic of the stage of the carcinoma.

The information thus derived is useful in prognosis and diagnosis,including susceptibility to acceleration of disease, status of adiseased state and response to changes in the environment, such as thepassage of time, treatment with drugs or other modalities. The cells canalso be classified as to their ability to respond to therapeutic agentsand treatments, isolated for research purposes, screened for geneexpression, and the like. The clinical samples can be furthercharacterized by genetic analysis, proteomics, cell surface staining, orother means, in order to determine the presence of markers that areuseful in classification. For example, genetic abnormalities can becausative of disease susceptibility or drug responsiveness, or can belinked to such phenotypes.

Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims. In this specification andthe appended claims, the singular forms “a,” “an” and “the” includeplural reference unless the context clearly dictates otherwise.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing the subject components ofthe invention that are described in the publications, which componentsmight be used in connection with the presently described invention.

As summarized above, the subject invention is directed to methods ofclassification of cancers, as well as reagents and kits for use inpracticing the subject methods. The methods may also determine anappropriate level of treatment for a particular cancer.

Methods are also provided for optimizing therapy, by firstclassification, and based on that information, selecting the appropriatetherapy, dose, treatment modality, etc. which optimizes the differentialbetween delivery of an anti-proliferative treatment to the undesirabletarget cells, while minimizing undesirable toxicity. The treatment isoptimized by selection for a treatment that minimizes undesirabletoxicity, while providing for effective anti-proliferative activity.

The invention finds use in the prevention, treatment, detection orresearch transitional cell carcinomas. Carcinomas are malignancies thatoriginate in the epithelial tissues. Epithelial cells cover the externalsurface of the body, line the internal cavities, and form the lining ofglandular tissues. In adults, carcinomas are the most common forms ofcancer. The urinary bladder is lined by “transitional cells.” Atransitional cell carcinoma is a tumor of the transitional cell liningof the urinary bladder.

“Diagnosis” as used herein generally includes determination of asubject's susceptibility to a disease or disorder, determination as towhether a subject is presently affected by a disease or disorder,prognosis of a subject affected by a disease or disorder (e.g.,identification of pre-metastatic or metastatic cancerous states, stagesof cancer, or responsiveness of cancer to therapy), and use oftherametrics (e.g., monitoring a subject's condition to provideinformation as to the effect or efficacy of therapy).

The term “biological sample” encompasses a variety of sample typesobtained from an organism and can be used in a diagnostic or monitoringassay. The term encompasses blood and other liquid samples of biologicalorigin, solid tissue samples, such as a biopsy specimen or tissuecultures or cells derived therefrom and the progeny thereof. The termencompasses samples that have been manipulated in any way after theirprocurement, such as by treatment with reagents, solubilization, orenrichment for certain components. The term encompasses a clinicalsample, and also includes cells in cell culture, cell supernatants, celllysates, serum, plasma, biological fluids, and tissue samples.

The terms “treatment”, “treating”, “treat” and the like are used hereinto generally refer to obtaining a desired pharmacologic and/orphysiologic effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of a partial or complete stabilization orcure for a disease and/or adverse effect attributable to the disease.“Treatment” as used herein covers any treatment of a disease in amammal, particularly a human, and includes: (a) preventing the diseaseor symptom from occurring in a subject which may be predisposed to thedisease or symptom but has not yet been diagnosed as having it; (b)inhibiting the disease symptom, i.e., arresting its development; or (c)relieving the disease symptom, i.e., causing regression of the diseaseor symptom.

The terms “individual,” “subject,” “host,” and “patient,” usedinterchangeably herein and refer to any mammalian subject for whomdiagnosis, treatment, or therapy is desired, particularly humans. Othersubjects may include cattle, dogs, cats, guinea pigs, rabbits, rats,mice, horses, and the like.

A “host cell”, as used herein, refers to a microorganism or a eukaryoticcell or cell line cultured as a unicellular entity which can be, or hasbeen, used as a recipient for a recombinant vector or other transferpolynucleotides, and include the progeny of the original cell which hasbeen transfected. It is understood that the progeny of a single cell maynot necessarily be completely identical in morphology or in genomic ortotal DNA complement as the original parent, due to natural, accidental,or deliberate mutation.

The term “normal” as used in the context of “normal cell,” is meant torefer to a cell of an untransformed phenotype or exhibiting a morphologyof a non-transformed cell of the tissue type being examined.

“Cancerous phenotype” generally refers to any of a variety of biologicalphenomena that are characteristic of a cancerous cell, which phenomenacan vary with the type of cancer. The cancerous phenotype is generallyidentified by abnormalities in, for example, cell growth orproliferation (e.g., uncontrolled growth or proliferation), regulationof the cell cycle, cell mobility, cell-cell interaction, or metastasis,etc.

“Therapeutic target” generally refers to a gene or gene product that,upon modulation of its activity (e.g., by modulation of expression,biological activity, and the like), can provide for modulation of thecancerous phenotype.

As used throughout, “modulation” is meant to refer to an increase or adecrease in the indicated phenomenon (e.g., modulation of a biologicalactivity refers to an increase in a biological activity or a decrease ina biological activity).

Characterization of Transitional Cell Carcinoma Stem Cells

In transitional cell carcinomas, characterization of cancer stem cellsallows for the development of new treatments that are specificallytargeted against this critical population of cells, particularly theirability to self-renew, resulting in more effective therapies.“Transitional cell carcinomas”, as used herein, refers to the epithelialtumors

In human transitional cell carcinomas it is shown herein that there is asubpopulation of tumorigenic cancer cells with both self-renewal anddifferentiation capacity. These tumorigenic cells are responsible fortumor maintenance, and also give rise to large numbers of abnormallydifferentiating progeny that are not tumorigenic, thus meeting thecriteria of cancer stem cells. All tumorigenic potential was containedwithin the CD44⁺ subpopulation of cancer cells, which cells are furtherassociated with CK5 expression and activation of a self-renewal pathway.These cells were able to initiate tumor growth at a dose of from about10² cells, about 5×10² cells, about 10³ cells, providing at least a 100fold increase in tumor initiating potential compared to the CD44negative tumor cells.

The TCCSC are identified by their phenotype with respect to particularmarkers, and/or by their functional phenotype. In some embodiments, theTCCSC are identified and/or isolated by binding to the cell withreagents specific for the markers of interest. The cells to be analyzedmay be viable cells, or may be fixed or embedded cells.

In some embodiments, the reagents specific for the markers of interestare antibodies, which may be directly or indirectly labeled. Suchantibodies will usually include antibodies specific for CD44; and mayinclude antibodies specific for one or more of β-catenin; Stat3; orBmi-1.

Transitional Cell Carcinomas

In the US, >60,000 new cases of bladder cancer and about 12,700 deathsoccur each year. Bladder cancer is the 4th most common cancer among menand is less common among women; male:female incidence is about 3:1.Bladder cancer is more common among whites than blacks, and incidenceincreases with age. In >40% of patients, tumors recur at the same oranother site in the bladder, particularly if tumors are large, poorlydifferentiated, or multiple. Expression of tumor gene p53 may beassociated with progression.

More than 90% of bladder cancers are transitional cell carcinomas. Mostare papillary carcinomas, which tend to be superficial andwell-differentiated and to grow outward; sessile tumors are moreinsidious, tending to invade early and metastasize. Squamous cellcarcinoma is less common and usually occurs in patients with parasiticbladder infestation or chronic mucosal irritation. Adenocarcinoma mayoccur as a primary tumor but may reflect metastasis from intestinalcarcinoma, which should be ruled out. Bladder cancer tends tometastasize to the lymph nodes, lungs, liver, and bone. In the bladder,carcinoma in situ is high grade but noninvasive and usually multifocal;it tends to recur.

Most patients present with unexplained hematuria (gross or microscopic).Some present with anemia, and hematuria is detected during evaluation.Irritative voiding symptoms (dysuria, burning, frequency) and pyuria arealso common at presentation. Pelvic pain occurs with advanced cancer,when a pelvic mass may be palpable. Superficial bladder cancer rarelycauses death. For patients with deep invasion of the bladdermusculature, the 5-yr survival rate is about 50%, but adjuvantchemotherapy may improve these results. Generally, prognosis forpatients with progressive or recurrent invasive bladder cancer is poor.

Differential Cell Analysis

The presence of TCCSC in a patient sample can be indicative of the stageof the carcinoma. In addition, detection of TCCSC can be used to monitorresponse to therapy and to aid in prognosis. The presence of TCCSC canbe determined by quantitating the cells having the phenotype of the stemcell. In addition to cell surface phenotyping, it may be useful toquantitate the cells in a sample that have a “stem cell” character,which may be determined by the nuclear localization of one or more ofβ-catenin; Stat3; or Bmi-1, or by functional criteria, such as theability to self-renew, to give rise to tumors in vivo, e.g. in axenograft model, and the like.

Clinical samples for use in the methods of the invention may be obtainedfrom a variety of sources, particularly biopsy samples of transitionalcell carcinomas from patients, although in some instances samples suchas bone marrow, lymph, cerebrospinal fluid; synovial fluid, and the likemay be used. For analysis by histology methods, sections, which may befrozen, embedded, etc. are taken from a tumor sample. Once a sample isobtained, it can be used directly, frozen, or maintained in appropriateculture medium for short periods of time. The samples may be obtained byany convenient procedure. Typically the samples will be from humanpatients, although animal models may find use, e.g. equine, bovine,porcine, canine, feline, rodent, e.g. mice, rats, hamster, primate, etc.In some embodiments, where analysis by flow cytometry is desired, thetissue sample is dissociated, and the cell suspension may be separatedby centrifugation, elutriation, density gradient separation, apheresis,affinity selection, panning, FACS, centrifugation with Hypaque, etc.prior to analysis.

The cell sample is contacted with reagents specific for markers thatidentify TCCSC, as described above. The labeled cells are quantitated asto the expression of cell markers. A number of such methods are known inthe art.

The comparison of a differential progenitor analysis obtained from apatient sample, and a reference differential progenitor analysis isaccomplished by the use of suitable deduction protocols, AI systems,statistical comparisons, etc. A comparison with a reference tissueanalysis from normal cells, cells from similarly diseased tissue, andthe like, can provide an indication of the disease staging. A databaseof reference tissue analyses can be compiled. The methods of theinvention provide detection of a predisposition to more aggressive tumorgrow growth prior to onset of clinical symptoms, and therefore allowearly therapeutic intervention, e.g. initiation of chemotherapy,increase of chemotherapy dose, changing selection of chemotherapeuticdrug, and the like.

Cell Staining Methods

Analysis by cell staining may use conventional methods, as known in theart. Techniques providing accurate enumeration include confocalmicroscopy, fluorescence microscopy, fluorescence activated cellsorters, which can have varying degrees of sophistication, such asmultiple color channels, low angle and obtuse light scattering detectingchannels, impedance channels, etc. The cells may be selected againstdead cells by employing dyes associated with dead cells (e.g. propidiumiodide).

The affinity reagents may be specific receptors or ligands for the cellsurface molecules indicated above. In addition to antibody reagents,polynucleotide probes specific for an mRNA of interest, peptide-MHCantigen and T cell receptor pairs may be used; peptide ligands andreceptor; effector and receptor molecules, and the like. Antibodies andT cell receptors may be monoclonal or polyclonal, and may be produced bytransgenic animals, immunized animals, immortalized human or animalB-cells, cells transfected with DNA vectors encoding the antibody or Tcell receptor, etc. The details of the preparation of antibodies andtheir suitability for use as specific binding members are well-known tothose skilled in the art.

Of particular interest is the use of antibodies as affinity reagents.Conveniently, these antibodies are conjugated with a label for use inseparation. Labels include magnetic beads, which allow for directseparation, biotin, which can be removed with avidin or streptavidinbound to a support, fluorochromes, which can be used with a fluorescenceactivated cell sorter, or the like, to allow for ease of separation ofthe particular cell type. Fluorochromes that find use includephycobiliproteins, e.g. phycoerythrin and allophycocyanins, fluoresceinand Texas red. Frequently each antibody is labeled with a differentfluorochrome, to permit independent sorting for each marker.

The antibodies are added to cells, and incubated for a period of timesufficient to bind the available antigens. The incubation will usuallybe at least about 5 minutes and usually less than about 30 minutes. Itis desirable to have a sufficient concentration of antibodies in thereaction mixture, such that the efficiency of the separation is notlimited by lack of antibody. The appropriate concentration is determinedby titration. The medium in which the cells are separated will be anymedium that maintains the viability of the cells. A preferred medium isphosphate buffered saline containing from 0.1 to 0.5% BSA. Various mediaare commercially available and may be used according to the nature ofthe cells, including Dulbecco's Modified Eagle Medium (dMEM), Hank'sBasic Salt Solution (HBSS), Dulbecco's phosphate buffered saline (dPBS),RPMI, Iscove's medium, PBS with 5 mM EDTA, etc., frequently supplementedwith fetal calf serum, BSA, HSA, etc.

Analysis may be performed based on in situ hybridization analysis, orantibody binding to tissue sections. Such analysis allows identificationof histologically distinct cells within a tumor mass, and theidentification of genes expressed in such cells. Sections forhybridization may comprise one or multiple solid tumor samples, e.g.using a tissue microarray (see, for example, West and van de Rijn (2006)Histopathology 48 (1):22-31; and Montgomery et al. (2005) ApplImmunohistochem Mol Morphol. 13 (1):80-4). Tissue microarrays (TMAs)comprise Multiple sections. A selected probe, e.g. antibody specific fora marker of interest; or probe specific for β-catenin, is detectablelabeled, and allowed to bind to the tissue section, using methods knownin the art. The staining may be combined with other histochemical orimmunohistochemical methods. The expression of selected genes in astromal component of a tumor allows for characterization of the cellsaccording to similarity to a stromal cell correlate of a soft tissuetumor.

The labeled cells are then analyzed as to the expression of cell surfacemarkers as previously described.

TCCSC Compositions

The cells of interest may be separated from a complex mixture of cellsby techniques that enrich for cells having the above describedcharacteristics. For isolation of cells from tissue, an appropriatesolution may be used for dispersion or suspension. Such solution willgenerally be a balanced salt solution, e.g. normal saline, PBS, Hank'sbalanced salt solution, etc., conveniently supplemented with fetal calfserum or other naturally occurring factors, in conjunction with anacceptable buffer at low concentration, generally from 5-25 mM.Convenient buffers include HEPES, phosphate buffers, lactate buffers,etc.

The separated cells may be collected in any appropriate medium thatmaintains the viability of the cells, usually having a cushion of serumat the bottom of the collection tube. Various media are commerciallyavailable and may be used according to the nature of the cells,including dMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., frequentlysupplemented with fetal calf serum.

Compositions highly enriched for TCCSC are achieved in this manner. Thesubject population may be at or about 50% or more of the cellcomposition, and preferably be at or about 75% or more of the cellcomposition, and may be 90% or more. The desired cells are identified bytheir surface phenotype, by the ability to self-renew, ability to formtumors, etc. The enriched cell population may be used immediately, ormay be frozen at liquid nitrogen temperatures and stored for longperiods of time, being thawed and capable of being reused. The cellswill usually be stored in 10% DMSO, 50% FCS, 40% RPMI 1640 medium. Thepopulation of cells enriched for TCCSC may be used in a variety ofscreening assays and cultures, as described below.

The enriched TCCSC population may be grown in vitro under variousculture conditions. Culture medium may be liquid or semi-solid, e.g.containing agar, methylcellulose, etc. The cell population may beconveniently suspended in an appropriate nutrient medium, such asIscove's modified DMEM or RPMI-1640, normally supplemented with fetalcalf serum (about 5-10%), L-glutamine, a thiol, particularly2-mercaptoethanol, and antibiotics, e.g. penicillin and streptomycin.

The culture may contain growth factors to which the cells areresponsive. Growth factors, as defined herein, are molecules capable ofpromoting survival, growth and/or differentiation of cells, either inculture or in the intact tissue, through specific effects on atransmembrane receptor. Growth factors include polypeptides andnon-polypeptide factors. A wide variety of growth factors may be used inculturing the cells, e.g. LIF, steel factor (c-kit ligand), EGF,insulin, IGF, Flk-2 ligand, IL-11, IL-3, GM-CSF, erythropoietin,thrombopoietin, etc

In addition to, or instead of growth factors, the subject cells may begrown in a co-culture with fibroblasts, stromal or other feeder layercells. Stromal cells suitable for use in the growth of hematopoieticcells are known in the art. These include bone marrow stroma as used in“Whitlock-Witte” (Whitlock et al. [1985] Annu Rev Immunol 3:213-235) or“Dexter” culture conditions (Dexter et al. [1977] J Exp Med145:1612-1616); and heterogeneous thymic stromal

Screening Assays

TCCSC are also useful for in vitro assays and screening to detectfactors and chemotherapeutic agents that are active on cancer stemcells. Of particular interest are screening assays for agents that areactive on human cells. A wide variety of assays may be used for thispurpose, including immunoassays for protein binding; determination ofcell growth, differentiation and functional activity; production offactors; and the like.

In screening assays for biologically active agents, anti-proliferativedrugs, etc. the TCCSC composition, usually a culture comprising TCCSC,is contacted with the agent of interest, and the effect of the agentassessed by monitoring output parameters, such as expression of markers,cell viability, and the like. The cells may be freshly isolated,cultured, genetically altered, and the like. The cells may beenvironmentally induced variants of clonal cultures: e.g. split intoindependent cultures and grown under distinct conditions, for examplewith or without drugs; in the presence or absence of cytokines orcombinations thereof. The manner in which cells respond to an agent,particularly a pharmacologic agent, including the timing of responses,is an important reflection of the physiologic state of the cell.

Parameters are quantifiable components of cells, particularly componentsthat can be accurately measured, desirably in a high throughput system.A parameter can be any cell component or cell product including cellsurface determinant, receptor, protein or conformational orposttranslational modification thereof, lipid, carbohydrate, organic orinorganic molecule, nucleic acid, e.g. mRNA, DNA, etc. or a portionderived from such a cell component or combinations thereof. While mostparameters will provide a quantitative readout, in some instances asemi-quantitative or qualitative result will be acceptable. Readouts mayinclude a single determined value, or may include mean, median value orthe variance, etc. Characteristically a range of parameter readoutvalues will be obtained for each parameter from a multiplicity of thesame assays. Variability is expected and a range of values for each ofthe set of test parameters will be obtained using standard statisticalmethods with a common statistical method used to provide single values.

Agents of interest for screening include known and unknown compoundsthat encompass numerous chemical classes, primarily organic molecules,which may include organometallic molecules, inorganic molecules, geneticsequences, etc. An important aspect of the invention is to evaluatecandidate drugs, including toxicity testing; and the like.

In addition to complex biological agents candidate agents includeorganic molecules comprising functional groups necessary for structuralinteractions, particularly hydrogen bonding, and typically include atleast an amine, carbonyl, hydroxyl or carboxyl group, frequently atleast two of the functional chemical groups. The candidate agents oftencomprise cyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Candidate agents are also found among biomolecules,including peptides, polynucleotides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof.

Included are pharmacologically active drugs, genetically activemolecules, etc. Compounds of interest include chemotherapeutic agents,hormones or hormone antagonists, etc. Exemplary of pharmaceutical agentssuitable for this invention are those described in, “The PharmacologicalBasis of Therapeutics,” Goodman and Gilman, McGraw-Hill, New York, N.Y.,(1996), Ninth edition, under the sections: Water, Salts and Ions; DrugsAffecting Renal Function and Electrolyte Metabolism; Drugs AffectingGastrointestinal Function; Chemotherapy of Microbial Diseases;Chemotherapy of Neoplastic Diseases; Drugs Acting on Blood-Formingorgans; Hormones and Hormone Antagonists; Vitamins, Dermatology; andToxicology, all incorporated herein by reference. Also included aretoxins, and biological and chemical warfare agents, for example seeSomani, S. M. (Ed.), “Chemical Warfare Agents,” Academic Press, NewYork, 1992).

Test compounds include all of the classes of molecules described above,and may further comprise samples of unknown content. Of interest arecomplex mixtures of naturally occurring compounds derived from naturalsources such as plants. While many samples will comprise compounds insolution, solid samples that can be dissolved in a suitable solvent mayalso be assayed. Samples of interest include environmental samples, e.g.ground water, sea water, mining waste, etc.; biological samples, e.g.lysates prepared from crops, tissue samples, etc.; manufacturingsamples, e.g. time course during preparation of pharmaceuticals; as wellas libraries of compounds prepared for analysis; and the like. Samplesof interest include compounds being assessed for potential therapeuticvalue, i.e. drug candidates.

The term “samples” also includes the fluids described above to whichadditional components have been added, for example components thataffect the ionic strength, pH, total protein concentration, etc. Inaddition, the samples may be treated to achieve at least partialfractionation or concentration. Biological samples may be stored if careis taken to reduce degradation of the compound, e.g. under nitrogen,frozen, or a combination thereof. The volume of sample used issufficient to allow for measurable detection, usually from about 0.1:Ito 1 ml of a biological sample is sufficient.

Compounds, including candidate agents, are obtained from a wide varietyof sources including libraries of synthetic or natural compounds. Forexample, numerous means are available for random and directed synthesisof a wide variety of organic compounds, including biomolecules,including expression of randomized oligonucleotides and oligopeptides.Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant and animal extracts are available or readily produced.Additionally, natural or synthetically produced libraries and compoundsare readily modified through conventional chemical, physical andbiochemical means, and may be used to produce combinatorial libraries.Known pharmacological agents may be subjected to directed or randomchemical modifications, such as acylation, alkylation, esterification,amidification, etc. to produce structural analogs.

Agents are screened for biological activity by adding the agent to atleast one and usually a plurality of cell samples, usually inconjunction with cells lacking the agent. The change in parameters inresponse to the agent is measured, and the result evaluated bycomparison to reference cultures, e.g. in the presence and absence ofthe agent, obtained with other agents, etc.

The agents are conveniently added in solution, or readily soluble form,to the medium of cells in culture. The agents may be added in aflow-through system, as a stream, intermittent or continuous, oralternatively, adding a bolus of the compound, singly or incrementally,to an otherwise static solution. In a flow-through system, two fluidsare used, where one is a physiologically neutral solution, and the otheris the same solution with the test compound added. The first fluid ispassed over the cells, followed by the second. In a single solutionmethod, a bolus of the test compound is added to the volume of mediumsurrounding the cells. The overall concentrations of the components ofthe culture medium should not change significantly with the addition ofthe bolus, or between the two solutions in a flow through method.

Preferred agent formulations do not include additional components, suchas preservatives, that may have a significant effect on the overallformulation. Thus preferred formulations consist essentially of abiologically active compound and a physiologically acceptable carrier,e.g. water, ethanol, DMSO, etc. However, if a compound is liquid withouta solvent, the formulation may consist essentially of the compounditself.

A plurality of assays may be run in parallel with different agentconcentrations to obtain a differential response to the variousconcentrations. As known in the art, determining the effectiveconcentration of an agent typically uses a range of concentrationsresulting from 1:10, or other log scale, dilutions. The concentrationsmay be further refined with a second series of dilutions, if necessary.Typically, one of these concentrations serves as a negative control,i.e. at zero concentration or below the level of detection of the agentor at or below the concentration of agent that does not give adetectable change in the phenotype.

Various methods can be utilized for quantifying the presence of theselected markers. For measuring the amount of a molecule that ispresent, a convenient method is to label a molecule with a detectablemoiety, which may be fluorescent, luminescent, radioactive,enzymatically active, etc., particularly a molecule specific for bindingto the parameter with high affinity. Fluorescent moieties are readilyavailable for labeling virtually any biomolecule, structure, or celltype. Immunofluorescent moieties can be directed to bind not only tospecific proteins but also specific conformations, cleavage products, orsite modifications like phosphorylation. Individual peptides andproteins can be engineered to autofluoresce, e.g. by expressing them asgreen fluorescent protein chimeras inside cells (for a review see Joneset al. (1999) Trends Biotechnol. 17 (12):477-81). Thus, antibodies canbe genetically modified to provide a fluorescent dye as part of theirstructure. Depending upon the label chosen, parameters may be measuredusing other than fluorescent labels, using such immunoassay techniquesas radioimmunoassay (RIA) or enzyme linked immunosorbance assay (ELISA),homogeneous enzyme immunoassays, and related non-enzymatic techniques.The quantitation of nucleic acids, especially messenger RNAs, is also ofinterest as a parameter. These can be measured by hybridizationtechniques that depend on the sequence of nucleic acid nucleotides.Techniques include polymerase chain reaction methods as well as genearray techniques. See Current Protocols in Molecular Biology, Ausubel etal., eds, John Wiley & Sons, New York, N.Y., 2000; Freeman et al. (1999)Biotechniques 26 (1):112-225; Kawamoto et al. (1999) Genome Res 9(12):1305-12; and Chen et al. (1998) Genomics 51 (3):313-24, forexamples.

Kits may be provided, where the kit will comprise staining reagents thatare sufficient to differentially identify the TCCSC described herein. Amarker combination of interest may include CD44 and a lineage panel asdescribed herein. In other embodiments, a probe or antibody specific forBmi1 may be included. The staining reagents are preferably antibodies,and may be detectably labeled. Kits may also include tubes, buffers,etc., and instructions for use.

Each publication cited in this specification is hereby incorporated byreference in its entirety for all purposes.

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,and reagents described, as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which will be limited only by the appendedclaims.

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the culture” includes reference to one or more culturesand equivalents thereof known to those skilled in the art, and so forth.All technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs unless clearly indicated otherwise.

EXPERIMENTAL

Bladder cancer is the second most common urologic malignancy and isincurable at its advanced stage. Evidence is emerging to support thathuman tumors contain a rare population of cancer stem cells (CSCs),which are best defined functionally by their unique biologicalproperties, including tumor-initiating potential, self-renewal anddifferentiation abilities. In the current study, we identify for thefirst time a CSC population in an advanced stage patient bladder cancer.In 6 out of 8 patient bladder cancers, we identified a rare populationof CD44⁺ tumor cells, comprising ˜1-10% of total tumor population. In axenograft model, as few as 100 CD44⁺ tumor cells was able to generatetumors, and was at least 200 fold enriched for tumor-initiatingpotential in a limiting dilution analysis when compared to CD44⁻ tumorcells. Further, CD44⁺ tumor cells were able to generate seriallytransplantable xenograft tumors (indicative of self-renewal) thatrecapitulate the heterogeneity of original tumor (differentiation)indicated by FACS and histology. Immunofluorescence analysis usingconfocal microscopy demonstrated nuclear localization of β-catenin, oractivation of this self-renewal signaling pathway in ˜30-70% of CD44⁺tumor cells. In conclusion, these data demonstrate proof of principalfor the existence of a tumor-initiating cell population with stemcell-like properties in bladder cancer.

TABLE 1 Frequency of TCC tumor cell engraftment from CD44 positive andnegative fractions in immunocompromised mice. CD44⁺ CD44⁻ unsortedPrimary patient tumor 2.5 × 10⁵ cells 1/1 0/1 — Mouse 1^(st) passage 1.0× 10⁵ cells 2/2 ½ — Mouse 2^(nd) passage 5.0 × 10⁶ cells — — 1/1 1.0 ×10⁶ cells — — 1/1 5.0 × 10⁵ cells — — 1/1 2.5 × 10⁵ cells — 3/3 1/1 1.0× 10⁵ cells 3/3 ⅓ 0/1 5.0 × 10⁴ cells 4/4 2/4 — 2.0 × 10⁴ cells 4/4 ¼ —5.0 × 10³ cells 4/4 0/4 —   1000 cells 2/4 0/4 —    100 cells ¼ 0/4 —

Example 2 Molecular Profiling of Patient Bladder Cancers RevealsHeterogeneity in Active Self-Renewing Pathways and the Existence ofUnique Tumor-Initiating Cells

Bladder cancer is a heterogeneous disease. Enzymatically dissociatedpatient bladder transitional cell carcinomas (TCCs) formed xenografts inthe skin of immunocompromised mice; engraftment success closelycorrelated with their TNM staging. Through screening bladder TCCsuspensions with CD44, CD133, CD24, CD49f, epithelial specific antigen,CD166 and CD105, we found a heterogeneity in the immunophenotype of theTCCs. CD44 was consistently expressed in 13 out of 14 bladder TCCsanalyzed as a small subpopulation (3.4 to 36.3%) of total tumor cells.Five out of 13 bladder TCCs engrafted in vivo, and the CD44+subpopulation consistently enriches for a tumor-initiating population;this CD44+ subset is 10-200 fold more tumorigenic than CD44− cellswithin the same tumor. We analyzed a tissue array containing more than300 bladder TCCs by immunohistochemistry. Approximately 40.4% of TCCscontain CD44+ cells; these cells usually express cytokeratin 5 (CK5)(P<0.0001) but not cytokeratin 20 (P=0.8160). Further, molecularprofiling of CD44+ bladder TCCs focusing on oncogenic pathways that werealso implicated in the self-renewal of stem cells (i.e. β-catenin,Bmi-1, Stat3, Oct4 and Nanog) classifies tumors into subgroups. Asignificant correlation was found between CD44, Bmi-1 and Stat3 in themuscle invasive properties of bladder TCCs (P=0.0047). In one TCC, CD44+tumor-initiating cells expressed nuclear, activated β-catenin. Thesedata revealed a heterogeneity in bladder TCCs; rare tumor-initiatingcells likely drive tumor development through various oncogenic pathwaysin different subset of patients.

Results

Establishment of a xenograft model with engraftment ability closelyassociating with the TNM staging of original patients. We have collected13 freshly isolated patient bladder TCCs, eleven of which are muscleinvasive (pT2 and pT3 stage), one of which is carcinoma in situ and onewithout disclosed clinical information (Table 2). Tumors weredissociated into viable cell suspensions by enzymatic digestion, and wehave tested their relative ability to engraft in an immunocompromisedmouse strain that is deficient in recombinase activating gene 2 (RAG2)and the common cytokine receptor γ chain (γc) (RAG2⁻/γc⁻). These micelack of T, B and NK cells and were more efficient in human primary cellengraftment. There was only a 35.7% (5 out of 14) successful tumor takerate with usually three months latency when tumor cells were injectedintradermally. Importantly, the relative ability for tumors to engraftin vivo seemed to associate closely with the TNM staging of originalpatients (Table 2), with 4 out of 6 pT3 stage tumors engrafted, 1 out of5 pT2 stage tumors engrafted and none engrafted below pTa stage. In ahigh percentage of mice that were engrafted with human tumors (3 out of5) immunocompromised mice died early due to pneumocystis pneumoniaand/or bacterial bronchopneumonia. Of the two tumors that were seriallytransplantable in vivo, both were at pT3 stages (Table 2). Inparticular, original patient tumor #8 with lymph node metastasis (pT3apN1 pMX) (FIG. 6C) also formed lymph node metastasis after 8 months ofengraftment in immunocompromised mice (FIGS. 6D, E & F), withhistopathology closely resembling the original patient tumor (FIG.6A-F). Histological analysis by hematoxylin and eosin (H&E) stainingrevealed that xenograft tumors (FIG. 6B) often retain the originalhistology of patient tumors (FIG. 6A), an important criteria of axenograft model; suggesting specific subpopulation of patient tumorcells are not being selected in vivo.

Flow cytometry analysis of patient bladder TCCs reveals heterogeneity ofimmunophenotype. In several types of solid tumors, a uniquesubpopulation of tumorigenic cells was identified based on their abilityto form xenografts in immunocompromised mice. The general approachinvolved the utilization of flow cytometry to analyze andsub-fractionate patient tumors that were dissociated into cellsuspension. In brief, a tumorigenic subpopulation can either be isolatedor enriched based on the immunophenotype of tumor cells; for instance,CD44+/CD24−/ESA+ cells from patient breast tumors and pleural effusions,CD133+ tumor cells from glioblastomas and medullobastomas, CD44+ cellsfrom head and neck cancers, CD44+/CD166+ cells from colorectal cancersand CD44+/CD24+/ESA+ cells from pancreatic cancers were demonstrated tocontain a tumorigenic subpopulation. Here, we took the lead from thesestudies and have analyzed 14 freshly isolated bladder TCCs by flowcytometry. Tumor cell suspensions were obtained by enzymaticdissociation; and infiltrating hematopoietic and endothelial cells wereexcluded based on the expression of CD45 and CD31 respectively. Evenwith a limited number of stem cell surface markers that we havescreened, which included CD44, CD133, CD24, CD49f/integrinα6, ESA, CD166and CD105, it is clear that individual tumors are very heterogeneous intheir immunophenotype. Currently, there are not sufficient specimensbeing analyzed to define a clear association between relativeimmunophenotype and various TNM staging. Importantly, one single markerCD44 is consistently expressed in 13 out of 14 bladder tumors beinganalyzed, comprising an expression frequency from ˜3.4% to 36.3% of thetotal population (Table 2 & 3), while 1 out of 14 specimen did notcontain CD44 expression (Table 2).

In vivo human xenograft model reveals a unique tumor-initiating cellpopulation in high grade patient TCCs. Therefore, we decided to focus onthis marker and determine whether CD44 expressing bladder tumor cellscontain unique biological properties in vivo. To determine the relativetumorigenic potential in vivo compared to other tumor cells within thesame tumor, we purified the CD44+ subpopulation by flow cytometry andinjected them intradermally into RAG2⁻/γc⁻ mice. In 5 out of 14 patientTCC specimens, the fractionated CD44⁺ tumor cell subpopulation engraftedas xenograft, while CD44⁻ cells from the same tumor either do notengraft or require a relatively higher number of CD44⁻ cells to engraftin vivo (Table 3). These data revealed that a CD44⁺ tumor cellsubpopulation enriches for tumor-initiating cells from these 5 bladderTCCs. Further, in two of these engrafted CD44⁺ xenografts (patient #1 &8), we found that they were able to generate xenograft tumors uponserial transplantation. In the second serial transplantation, we wereable to perform detailed limiting dilution experiment on un-fractionatedand fractionated tumor cells. We found that at least 2.5×10⁵un-fractionated tumor cells were required to generate xenografts (Table3, patient #1). Further, we found that pure CD44⁺ tumor cells can formxenografts more effectively than CD44⁻ cells within the same tumor.Remarkably, as few as 100-500 CD44⁺ tumor cells can form xenografts invivo (Table 3, patient #1 & 8), suggesting a frequency of at least 1 outof 100-500 CSC was present in CD44⁺ bladder tumor cells; while at least2.0-5.0×10⁴ CD44⁻ tumor cells were necessary to induce xenografts (Table3, patient #1 & 8). These data clearly suggest that in seriallytransplanted xenografts, CD44⁺ bladder tumor cells also containtumor-initiating cells and were at least 100 to 200-fold enriched fortumor-initiating potential when compared to CD44⁻ cells (Table 3,patient #1 & 8).

CD44 positive tumor cells possess unlimited self-renewing capacity incomparison to CD44 negative tumor cells in pT3 stage bladder cancers. Inpatient TCC specimens #1 & 8, CD44⁺ tumor cells were able to generateserially transplantable xenograft tumors in RAG2⁻/γc⁻ mice. Flowcytometry analysis revealed that xenografts derived from pure patientCD44⁺ tumor cells (FIGS. 6H & K, Black box) gave rise to a heterogeneoustumor population containing both the basal cell-like CD44⁺ (FIGS. 6H &K, Black Box) and more differentiated CD44⁻ tumor cells (FIGS. 1H & K,Gray Box). Mouse cells were excluded from the analysis with a cocktailcontaining antibodies to mouse CD31 (endothelial), CD45 (hematopoieticcell) and H2K^(d) (mouse histocompatibility class I) molecules (FIGS. 6H& K, blue box). Furthermore, H&E staining of xenografts derived fromCD44⁺ tumor cells of patient #1 revealed areas of less differentiatedtumor cells (FIG. 6I, area indicated by asterix), as well as terminallydifferentiated tumor cells with high keratinization (FIG. 6J, areaindicated by arrows). On the other hand, in xenografts formed from arelatively higher number of CD44⁻ tumor cells (2-5×10⁴) from patient #1(FIG. 6H, Gray Box), flow cytometry revealed that the majority of cellsin the tumor were CD44⁻ cells. Histological analysis with H&E stainingin these xenograft tumors revealed areas mostly comprised of highlykeratinized, terminally differentiated cells (FIG. 6J, area indicated byarrows) with very few cellular components (FIG. 6J). For patient #8,xenografts formed from CD44+ and CD44− tumor cells did not demonstrate asignificant difference in histology, revealing pathological differencesbetween individual tumor specimens. Further, xenografts formed from ahigher number of CD44− cells (3.0×10⁵) reveal immunophenotype mostlycomprising of CD44− tumor cells (patient #8, FIG. 1G), importantly, ashigh as 1×10⁵ CD44− tumor cells from this xenograft line were not ableto generate secondary xenografts upon serial transplantation, indicatinga limited self-renewal and/or proliferative capacity of CD44− tumorcells.

Collectively, these data convincingly revealed that CD44⁺ tumor cells inbladder TCCs contain a “self-renewal” property, which can give rise tobasal-cell like CD44⁺ tumor cells; and retain the ability to“differentiate” into a more differentiated CD44⁻ tumor cell population.Although a higher number of CD44− tumor cells can form xenografts invivo, their limited self-renewal ability is revealed by their inabilityto form xenografts upon serial transplantation.

CD44 expressing subpopulation in TCC specimens retain cytokeratinexpression and cellular morphology and properties resembling that ofurothelial basal cells. In normal human bladder urothelium, it waspreviously reported that CD44 primarily localizes in the basal cellcompartment. We are interested to determine whether CD44+ bladder tumorcells retain properties similar to normal urothelial cells with theconventional basal cell marker cytokeratin 5 or differentiated cellmarker cytokeratin 20 by immunohistochemistry, in a tissue arraycontaining ˜337 bladder TCC specimens. Immunohistochemical data wasanalyzed and scored based on the percentage of positive expression foreach marker within individual tissue section. The bladder TCC specimenswere then clustered into subgroups based on the relative expression ofCD44, CK5 and CK20. The results were summarized in a colorimetricrepresentation as demonstrated in FIG. 7A. Red represents positivecases, green represents negative cases and grey represents data that aremissing. This bladder cancer tissue array contains 84 cases of femaleand 253 cases of male to a total of 337 patients in a 3:1 male to femaleratio. Approximately 59.6% (201 out of 337 cases) of the patient tissuesections stain negative for CD44 (represented in green), and 40.4% (136out of 337 cases) stain positive for CD44 (represented in red).Importantly, 84.6% of CD44 positive cases either stain positive for CK5(FIG. 8A, group 6 & 8) or do not stain positive for any cytokeratins(FIG. 8A, group 1). Multivariant regression analysis revealed astatistically significant correlation of CD44 and CK5 (P<0.0001)expression, but not with CK20 (P=0.8160). In bladder TCCs expressing allthree markers, i.e. CD44, CK5 and CK20 (group 8 as indicated by arrow),the CD44 expressing tumor cells often co-localize with CK5 and ismutually exclusive to CK20 in well differentiated, moderatelydifferentiated, as well as more dyplastic lesions (FIG. 7B).

Further, in limited cases of freshly isolated tumors (n=4), we were ableto examine the cellular morphology of CD44+ and CD44− cells preparedfrom cytospin, followed by Giemsa-Wright staining. As shown in FIG. 8Cin a representative TCC, CD44⁺ tumor cells possess a high nuclear tocytoplasmic (NC) ratio (from patient #3). In addition, CD44⁺ tumor cellswere relatively smaller and homogenous in size (FIG. 7C), typicalcharacteristics of basal urothelial cells. On the other hand, CD44⁻tumor cells were more heterogeneous in size, containing cells withcharacteristics of moderately and terminally differentiation (FIG. 7C).In other cases, either there weren't significant differences in cellularmorphologies between CD44+ and CD44− cells or follow similar morphologyof that described in FIG. 7C. Collectively, these data demonstrate thatCD44 expressing tumor cells retain cytokeratin markers and cellularmorphology similar to normal urothelial basal cells.

Subclassification of bladder TCCs by molecular profiling of CD44 andoncogenic pathways that are implicated in the self-renewal of stemcells. Further, we focused on these 40.4% of CD44 expressing tumorspecimens in the bladder cancer tissue array (described in FIG. 7), inattempt to identify signaling molecules that have important clinicalimplications for this subset of tumors. Certain signaling molecules suchβ-catenin, Bmi-1, Stat3, Oct-4 and Nanog have been implicated tomaintain self-renewal of adult or embryonic stem cells. Interestingly,some of these signaling molecules crucial for the self-renewal of stemcells are also implicated in the tumorigenic process.

Immunohistochemical analysis of these oncoproteins in bladder cancersrevealed a heterogeneous molecular profile in pathway activation, whichsubclassifies bladder cancers into various groups based on theexpression of CD44 and the active state of Stat3, Bmi-1, β-catenin,Oct-4 and Nanog (illustrated in FIG. 8A). Red represents positive cases,green represents negative cases and grey represents data that aremissing. Within the CD44 expressing bladder cancers, different subsetsof tumors seem to utilize either one or more of theseoncogenic/self-renewal pathways (FIG. 8A). For instance, 10% of CD44+tumors (14 out of 140) contain nuclear localization of Bmi-1, 5% ofCD44+ contain nuclear localization of β-catenin (7 out of 140), and 45%of CD44+ tumors contain nuclear localization of Stat3 (35 out of 140)(FIG. 8A). Fifteen percent of CD44+ tumors contain activation of two ofsuch oncogenic pathways being analyzed (21 out of 140). Importantly,there is a statistically significant correlation between CD44, Bmi-1 andStat3 in relation to the invasive properties of bladder cancer(P=0.0047). While activation of β-catenin has an association with highgrade bladder cancers, it is not statistically significant (P=0.06).Careful analysis revealed that although a large fraction of nuclearStat3 (FIG. 8B) or Bmi-1 (FIG. 8C) positive cells co-localize with CD44+tumor cells, their expression is not completely restricted CD44+ tumorcells. In contrast, in the relatively small subset of tumor specimenswith both CD44 expression and active/nuclear β-catenin, nuclearβ-catenin seemed to strictly localize within CD44+ tumor cells (FIG.8D).

On the other hand, we obtained no positive staining for Oct-4 and Nanogin all bladder cancer specimens analyzed (FIG. 8A, indicated in greencolor); although we were able to obtain strong nuclear staining of Oct-4(FIG. 8E) and Nanog (FIG. 8F) in patient seminomas as positive controland no background staining in normal testis as negative control (FIGS.8E & F). These data are in contrast to a recent report that activated ornuclear accumulation of Oct-4 is of high incidence, in 31 out of 32patient bladder tumors analyzed.

In the current study, we have employed multiple approaches to identify aCD44 positive tumorigenic subpopulation with unlimited self-renewalcapacity from bladder TCCs. This unique subpopulation retainscytokeratin markers and cellular morphologies resembling that ofurothelial basal cells. Importantly, molecular profiling of CD44positive bladder cancers revealed heterogeneity of self-renewalpathways. These data implicate that diverse sets of epigenetic andgenetic alterations can accumulate in urothelial basal cells fromdifferent patients, leading to this molecular heterogeneity that cansubclassify bladder TCCs. These data revealed significant clinicalimplications, that different subgroup of bladder TCC patients mayrespond to drugs that target different sets of signaling pathways.Therefore, the evaluation of successful therapeutics in current clinicaltrials based on the response of a major patient population may not beentirely valid, which may overlook agents that target a smaller subgroupof patients. Further, the existence of a tumorigenic subpopulation inbladder cancers suggest that current approaches in molecular profilingof bulk tumor populations may not reveal effective therapeutic targets.

Normal urothelium is a slow cycling epithelium, which turnovers every 6months to 1 year. However, in response to wounding induced by physicaland chemical injury, mouse and rat urothelium can initiate regenerationin as little as 12 hours, peak at 24 hours and completely heal over thewound site in 2-3 days. Further, the urothelium undergoes frequentsquamous metaplasia (when one differentiated cell type is replaced byanother), especially under a vitamin A deficient diet. This slow cyclingurothelium with high regenerative potential and high plasticity of theurothelium support the hypothesis that an adult cell progenitorpopulation exists within the adult bladder. The median age of bladdercancer at diagnosis in the United States is 63, and it is wellestablished that chronic exposure to cigarette smoking or occupationalexposure to chemicals such as polycyclic aromatic hydrocarbons (PAH) hasan established link to bladder cancer risk. Further, cancer patient whoreceived high dose treatment of the chemotherapeutic agentcyclophosphamide and/or irradiation also has a higher risk in developingbladder cancer. On other hand, chronic inflammation of the bladdercaused by a parasite Schistosoma hematobium in the region of NorthernAfrica also have a risk to the development of squamous cell carcinomas(SCCs) of the bladder, although over 90% of bladder cancers aretransitional cell carcinomas.

These evidences led to our hypothesis that there may be a “target cell”population within the normal urothelium, which preferentially accumulateepigenetic and genetic alterations caused by continued physical andchemical assaults from various sources, eventually leading to the lossof tissue homeostasis through extensive cell proliferation, block ofdifferentiation, insensitivity to apoptotic signals and/or genomicinstability after a long latency period (˜63 years). Our data revealed aCD44+ tumorigenic subpopulation with unlimited self-renewal capacity inbladder TCCs (Table 3 and FIG. 6). This subpopulation retainedcytokeratin markers and cellular morphology resembling that of normalurothelial basal cells (FIG. 7), indicating that the basal cellcompartment is the “target cell” population for transitional cellcarcinoma initiation.

Another important observation extending from the current study is themolecular heterogeneity of bladder TCCs, even from a small screenfocusing on oncogenic pathways that are also implicated in theself-renewal of stem cells. This raised the possibility that differentsubset of bladder TCC patients, although may contain the same “targetcell” population for tumor initiation, may indeed accumulate totallydiverse set of epigenetic and mutagenic alterations leading to thedevelopment of advanced stage disease. In CD44 expressing bladder TCCs,there is a significant correlation between CD44, Bmi-1 and Stat3 in theinvasive properties of this disease. Although long term clinical followup or survival data from this set of patients is currently unavailable,there is reported correlation between the invasive properties of bladderTCCs and poor prognosis, and invasion remained one of the hallmarks forthe classification (TNM staging), prognosis, and management of thisdisease. Our study is the first to reveal the possible role of oncogenicBmi-1 and Stat3 in the invasive properties of bladder TCCs. Currently,we are collecting patient specimens from such subgroups to evaluate thefunctional significance of these individual pathways in maintaining theinitiation and growth of xenograft tumors in vivo. In conclusion, ourcurrent data support the notion that unique tumor-initiating cells withunlimited self-renewal capacity exist in bladder TCCs, which drive tumordevelopment through different oncogenic pathways in diverse subset ofpatients.

Experimental Procedures

Bladder tumor tissue dissociation. Enrollment of human subjects to thecurrent study has been approved by the Stanford Institutional ReviewBoard under the protocol 1512. Individual consents have been obtainedfrom all subjects. Tumor tissues obtained from cystectomy or xenograftwere immediately incubated in calcium free Hanks' Balanced Salt Solution(Invitrogen, NY) with 2% chelexed fetal bovine serum (HBSS/2% FBS).Tumors were disaggregated mechanically with sterile scalpel and mincedinto 1-2 mm³ pieces, followed by enzymatic digestion in media 199containing proteolytic (Accumax, Innovative Cell Technologies, Inc.),collagenolytic (200 U Type 1 and 20 U Type IV collagenase)(Sigma-Aldrich C-0130, C-5138) and DNAse enzymes at 37° C. for 2 to 6hours. Cells were then filtered through a 100 μm nylon mesh and washedonce with calcium free HBSS/2% FBS.

Analysis and cell separation by flow cytometry. Tumor cell suspensionswere washed with HBSS/2% FBS, incubated with mouse Ig for 10 min toprevent nonspecific antibody binding, and stained with PE-conjugatedanti-CD44 (1:50, BD Pharmingen 550989) antibody for 15 min at 4° C. Forpatient specimens, a lineage cocktail containing Cy7-PE-conjugatedanti-CD45 (1:200, BD Pharmingen 557748) and biotin-conjugated anti-CD31(1:100, eBioscience 13-0319-82) antibodies were stained for 15 min at 4°C., followed by a wash with HBSS/2% FBS and secondary antibodyincubation with Strepavidin-conjugated pacific blue antibody (1:400,Molecular Probes S11222). For xenograft specimens, a lineage cocktailcontaining biotin-conjugated rat anti-mouse CD45 (1:200, BD Pharmingen553077), CD31 (1:200, BD Pharmingen 553371) and H2K^(d) (1:200, BDPharmingen 553564) antibodies were stained for 15 min at 4° C., followedby a wash with HBSS/2% FBS and secondary antibody incubation withStrepavidin-conjugated pacific blue antibody. Finally, tumor cellsuspensions were resuspended in 1 mg/ml propidium iodide (PI). Flowcytometry analysis and cell sorting was performed on FACSAria (BectonDickinson) under low pressure setting (20 psi) with a 100 μm nozzle.Data was analyzed using FlowJo software (Tree Star).

Transplantation of tumor cell suspension into immunocompromised mice.Either un-fractionated or fractionated tumor cell suspensions wereresuspended in high concentration matrigel (Becton Dickinson 354248).Adult Rag2⁻γc⁻ mice at age of 4-8 weeks were first shaved andanesthetized by 15 μl/g body weight of 2,2,2-Tribromoethanol(Sigma-Aldrich T-48402). Tumor cell suspensions were injectedintra-dermally into the dorsal side of mouse skin by 31 gauge insulinsyringes (Becton Dickinson). Tumor formation was monitored on a dailybasis.

Immunofluorescence staining and confocal microscopy. Tumor cells wereseparated by flow cytometry and resuspended in 10 μl of HBSS, placed onSuperfrost/Plus slides (Fisher Scientific), and semi-dry for 10-15 min.Cells were surrounded by ImmEdge™ pen (Vector Laboratories H-4000) andfixed with 0.2% paraformaldehyde (Electron Microscopy Sciences 15713-S)for 10 min. Cells were washed once with HBSS, non-specific antibodybinding was blocked with 10% goat serum for 15 min, and stained withanti-active-β-catenin antibody (Upstate 05-665 clone 8E7) at aconcentration of 1:200 for 30 min. Cells were then washed twice withHBSS and stained with goat anti-mouse AlexaFluor®594 (1:1000) or goatanti-mouse AlexaFluor®488 (1:500) secondary antibodies (MolecularProbes) for 30 min. Cells were washed twice with HBSS and slides weremounted with Vectorshield mounting media containing DAPI (VectorLaboratories H-1500). Slides were visualized under the Leica SP2 AOBSconfocal laser scanning microscope and images were collected with theLeica Confocal, v 2.5, build 1347 software.

Immunohistochemical analysis in tissue sections. Ten-micron paraffinembedded tissue sections were but, deparaffinized through 3 changes ofxylene 10 min each, and hydrated with graded ethanol (100% 2×, 95% 2×,80% 1× and 70% 1× with ddH₂O 2×). Antigen retrieval was performed with1M EDTA at pH8.0 (for anti-β-catenin) and 10 mM citrate buffer at pH6.0(all other antibodies) by microwave for 15 min. Slides were cooled forat least 30 min, and staining was performed using the DAKO EnVision kit(K4006 and K4011) following manufacture protocol. Primary antibodieswere applied at indicated concentrations, anti-β-catenin (BDTransduction 610154, 1:1000), anti-CD44 (BD Pharmingen 555477, 1:25),anti-Oct-3/4 (Santa Cruz sc-5279, 1:50) and anti-Bmi-1 (AbCam 14389-25,1:100). Slides were then counterstained with hematoxylin, dehydratedwith graded ethanol (95% 2×, 100% 3×), cleared by xylene and mounted inProtex mounting media. Five-micron frozen sections were stained with thesame protocol without going through the antigen retrieval step.

1-10. (canceled)
 11. A composition of mammalian transitional cellcarcinoma stem cells, wherein at least 50% of the cells in saidcomposition are CD44⁺ CK5⁺ transitional cell carcinoma stem cells(TCCSC).
 12. (canceled)
 13. The composition according to claim 11,wherein the TCCSC have increased nuclear localization of β-catenin;Stat3; or Bmi-1.
 14. (canceled)
 15. A method of screening a candidatechemotherapeutic agent for effectiveness against an TCCSC, the methodcomprising: contacting said agent with the cell composition of claim 11,and determining the effectiveness of said agent against said TCCSC. 16.The method of claim 15, further comprising classifying the TCCSCaccording to increased nuclear localization of β-catenin; Stat3; orBmi-1 prior to said contacting.